Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Cascade stacking and cascade electrofocusing: their interconversion and fundamental unity.

The composition of a stack [an isotachophoresis (ITP) system] containing multiple trailing buffer constituents (“cascade stack”) was computed using a modification of the program of Routs. On electrophoresis, such a buffer mixture gave rise to multiple moving boundaries in which either buffer constituents or proteins could be stacked. Buffer zones within the stack served as “spacers.” The cascade stack exhibits a pH gradient, sharp zone boundaries, and constant zone width irrespective of the duration of electrophoresis, just as in the case of a stack comprising a single leading and trailing constituent. The pH gradient, sharp zone boundaries, and the sequential order of protein zones were maintained when the cascade stack was transposed between strongly acidic and basic electrolytes. Such a transposed isotachophoretic gel functioned as an electrofocusing system, indistinguishable from electrofocusing gels made in either buffers (buffer electrofocusing, or BEF) or Ampholine (isoelectric focusing, or IF). In the converse experiment, a cascade electrofocusing gel, formed in the same buffer mixture used to form a cascade stack, was subjected to electrofocusing until the steady-state was attained and then it was transposed between the appropriate upper and lower buffers of the corresponding cascade stacking system. Such transposition gave rise to moving zones with the typically sharp boundaries of a stack, a transient state pH gradient, and an order of protein zones within the cascade stack identical to the cascade electrofocusing system. These studies indicate the essential physical-chemical identity between these two types of electrophoretic systems and indicate the need for continued development of a unified theory for isoelectric focusing and steady-state stacking (isotachophoresis).     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).     

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135–172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40–45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.     

Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient

A new method is described for preparative protein purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification never enters the Immobiline gel and thus does not have to be extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.     

固定pH梯度等电聚焦法分离纯化人胸腺活性肽

 本文用4—7月龄胎儿胸腺,按照匀浆—80℃加热—丙酮沉淀—抽提—超滤等一系列步骤制备人胸腺混合肽。用固定pH梯度聚丙烯酰胺凝胶等电聚焦技术,结合紫外薄层扫描法定位,从胸腺混合肽中得到四种纯化的胸腺肽HTP α_1,β_1,β_2和γ_1;分别测得其等电点为4.6,5.3,5.8和7.6,分子量分别为4800,6700,7200—7300和12,000—13,000。另外至少还得到四种部份纯化的胸腺肽。由玫瑰花结和末端转脱氧核苷酰酶活力测定结果,证明分离得到的各种胸腺肽均有较高的生物活性。     

Calf-ovary protein kinases dependent on adenosine 3':5' -monophosphate. Analysis by electrophoresis and electro-focusing on polyacrylamide get.

High resolving power and quantitative application polyacrylamide-gel electrophopresis at various pore sizes and electrofocusing provide resolution of a calf-ovarian protein-kinase system at an increased level of magnification, as well as optimal preparative routes. Three protein kinases dependent on adenosine 3':5' -monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5--10.2, appear to be aggregates of a commonsubmit or monomer. The three kinases are, by the criteria of polyacylamide gel electrophoresis, distinct from three adenosine-3':5' -monophosphate-binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins.     

Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients

We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (alpha + beta O2)2 and (alpha O2 beta +)2. The delta pI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).     

Isolation of Active cAMP Dependent Protein Kinases from Calf Ovaries: Gel Electrcphoresis vs. Gel Electrofocusting

Calf ovarian CAMP dependent protein kinase A was isolanted by adsorntion on to DEAF-cellulose, gel chromatography on agarosepolyacrylamide copolymer, electrophoresis in a 6% polyacrylamide gel, 0.2% in Iriton X-100, and DEAF-chromatography. The yield was 3.3 mg, representinw 22% of the starting material.

Purification was 400-fold. The product appears homogeneous on gel electropheresis at p 10.2, but DEAF-chromatography, gel elecectro focusing and gel electrophoresis at pH 8.5 and 7.5 reveal two charge isomeric forms of the enzyme.

Optimization of gel concentration for the separation of the enzyme from its closest migrating contaminant pointed to gel electrofocusing, rather than electrophoresis, as the appropriate Separation tool. However, that electrophoresis, as the inactivate the enzyme when conducted on wide-diameter preparative gels, if allowed to proceed to the steady-state, using either Ampholine or buffers as the carrier ampholytes, and etyleneglycol to repress no I yacrylami decopolyner, electrophorus is in a 6 po lyacrylaniide el, 0.7 in TM on -1 00, and DSAF-chromatography. The yield was 3.1 me, representing 787% of the starting material Petrification was 400-fold. The product appears homos jeneousonge I electrophoresis at pM 10.7, hut DEAF- chromatography, gel electron focus inn and ruels ectroohores is at pH 6.5 and 7.5 reveal two charred is on ericforirs of the enzyme, Opticalization of feel concentration for the separation of the enzyme from its closest ml floating cont eminent pointed to jel electro focusing, rather then elect rophoresis, as the appropriate separation tool. However, that method proved to inactivate the enzyme when c on ducted on wide-diameter preparative gels, if all owed to proceed to the steady-state, usinp; either Ampholine or buffers as the carrier ampholytes, and ethyl eneglycol to repress isoelectric precipitation. Only buffer electrofoucing on ultrogel Aca 54 if stoppert prior to the attainment of the isoelectric endpoint of the enzyme succeeded in recovering substantial (65%) activity, albeit at the price of resolution. Thus, a non-optimal concentration in polyacrylamide gel electrophoresis was applied in preference to preparative gel electro focusing.

Preparative methods for the isolation of Proter Kinase 8 and CAMP Binding Pritein A in homogeneous form were also developed, using nodifications of the above-stated procedure.     

Focusing of pepsin in strongly acidic immobilized pH gradients

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.     

Gradiflow as a prefractionation tool for two-dimensional electrophoresis

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions. Further, when the prefractionated acidic samples were analyzed on pH 4-7 immobilized pH gradient 2-D gels, improved resolution of the proteins within the chosen pH region was achieved compared to the unfractionated samples. This study demonstrates that the Gradiflow could be used as a preparative electrophoresis tool for the isolation of proteins into distinct pH fractions.     

An apparatus for immunoelectrofocusing. A study of squid tyrosinase.

An apparatus which facilitates immunoelectrofocusing is described. Polyacrylamide is used as the support medium for both the thin-layer isoelectric focusing and immunodiffusion stages. The pH gradients formed during electrofocusing are reproducible and sufficiently stable. Immunoelectrofocused and isoelectric focused patterns indicate that at least one inactive form and four active forms of squid tyrosinase exist.     

Preparative immobiline isoelectric focusing of plasma apolipoproteins on vertical slab gels

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis.

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of protein solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Stabilization of pH gradients in buffer electrofocusing on polyacrylamide gel.

pH gradients in buffer electrofocusing on polyacrylamide gel (BEF) have been stabilized for at least 100 hr, 25°C, by replacing the strongly acidic and basic anolyte and catholyte with isoelectric buffers identical to the terminal constituents of the pH gradient and gel. Such stabilization leads to a constant pI position of an electrofocused protein. No stabilization of pH gradients is achieved under otherwise identical conditions when strongly acidic and basic anolyte and catholyte are used.     

Phosphoglycolate phosphatase. Purification and properties.

Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis, and by isoelectric focusing. The highest specific activity obtained was 468 mumol of phosphate released/min/mg of protein. The native protein has a molecular weight of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 20,700, indicating the P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pI of 3.8 to 3.9 pH units by isoelectric focusing. Phosphosphoglycolate phosphatase from spinach leaves has a molecular weight of 93,000 and, unlike the enzyme from tobacco leaves, it is extremely unstable after DEAE-cellulose chromatography and is inactivated by lipase (EC 3.1.1.3). The phosphatase from both plants was stabilized by the addition of citrate or isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentration, while other phosphate esters of the photosynthetic carbon cycle were without effect.     

A Review of: “An Introduction to Separation Science B.L. Karger,L.R. Snyder and C. Horvath; Wiley-Interscience,New York, 1973; 586 pages; hard cover; $19.50”

The preparative buffer electrofocusing of a variety of thyroid related proteins is described. The study shows that selective electrofocusing of these proteins on granulated gels can be readily achieved with buffer systems of defined chemical composition. Specific proteins were recovered in satisfactory yields (30-70%) with sample loadings in the range 25-500mg.